Molecular Targets & Therapeutic Resistance Program

Overview | Leaders | Members | Research | Publications

Research Portfolio

Recent accomplishments of the MTTR Program include:

  • Identification of Caveolin-1 (CAV1) as an EWS/FLI1 target and a determinant of Ewing's sarcoma cells response to chemo- and radio-therapeutic treatments (Tirado et al., Cancer Res, 2006).
  • Identification of NF-kB (p65) as a target for radiosensitization that enhances the susceptibility of tumor cells to HDAC inhibition (Kim et al., Mol Cancer Ther, 2004; Kim et al., Carcinogenesis, 2005).
  • Completion of Phase I studies of liposomal c-Raf-1 antisense oligonucleotides against solid tumors, alone or in combination with radiation, in collaboration with Dr. John Marshall and members of the 3DT Program (Rudin et al., Clin Cancer Res, 2004; Dritschilo et al., Clin Cancer Res, 2006).
  • Identification of novel mechanism in the functional regulation of Base Excision Repair (BER) and Nucleotide Excision Repair (NER) pathways in mammalian cells (Adhikari et al., J Biol Chem, 2008).

View a full list of accomplishments from 2002 to 2008

Additionally, there are many ongoing studies:

Top

Targeting Ewing's sarcoma cell proliferation and response to chemoradiation
Investigators: Notario, Soldatenkov, Dritschilo

Caveolin-1 (CAV1) is an essential component of caveolae that is implicated in regulation of several signaling pathways and oncogenesis. Recently, CAV1 was shown to be a key tumorigenicity determinant for tumors of the Ewing's Sarcoma Family (ESFT). However, the possible role of CAV1 in the chemotherapy resistance commonly presented by an ESFT subset is unknown. These studies showed that CAV1 expression determines the sensitivity of Ewing's sarcoma (EWS) cells to chemotherapeutic agents currently used for ESFT treatment. Results from antisense- and short hairpin RNA-mediated gene expression knockdown, protein re-expression, and functional inhibitory studies demonstrated that CAV1 increases the resistance of EWS cells to doxorubicin (DXR)- and cisplatin (Cp)-induced apoptosis by a mechanism involving PKCα. CAV1 knockdown led to PKCα dephosphorylation and a concomitant sensitization to chemotherapy-induced apoptosis. Re-expression of CAV1 resulted in PKCα phosphorylation and decreased the sensitivity to DXR and Cp. Moreover, assays of reciprocal co-immunoprecipitation and immunofluorescence assays demonstrated co-localization of CAV1 and PKCα. These results identify CAV1 as a key determinant of the chemotherapy response of EWS cells, and imply that targeting CAV1 may allow the development of new molecular therapeutic strategies for ESFT patients.

Top

Differentiating DNA reactive vs. DNA non-reactive genotoxic mechanisms using gene expression profiles
Investigators: Fornace

The cellular stress response includes transcriptional changes leading to the activation of various physiological pathways involved in repair, survival and/or cell death. Recent progress in array technologies has enabled the monitoring of mRNA levels in large gene sets, or whole genomes, in response to toxic stimuli in a single experiment (toxicogenomics). The cellular stress response in mammalian cells is quite complex, involving hundreds and possibly thousands of genes. The published data indicate the utility of the gene expression profile analysis to provide insights into cellular physiological processes triggered by the treatment with genotoxicants. However, developing criteria for dose setting (e.g. a dose that induces a sufficient stress capable of producing a robust response at mRNA gene expression level at an early time-point) is critical when developing gene signatures with a goal to provide insights into genotoxic mechanisms. This toxicogenomics study resulted in the development of an experimental protocol and the construction of a reference database of gene expression profiles using agents with a variety of toxic mechanisms in the p53 proficient TK6 cell line. The gene expression profiles of individual agents in the database were in line with their molecular mechanisms of action. This platform will be utilized to evaluated the toxicity and elucidate the mechanism of action of target compounds.

Top

Studies on the role of poly (ADP-ribose) polymerase (PARP-1) on stress-induced transcription
Investigators: Dritschilo, Notario, Smulson, Jung, Soldatenkov

Agents that cause DNA strand breaks activate PARP, causing the post-translational modification of a variety of nuclear proteins involved in the response to DNA damage and repair. When damage leads to apoptotic cell death, PARP is proteolytically inactivated early in the process by the action of caspase-3. Pioneer studies by Drs. Dritschilo, Notario, and Smulson previously demonstrated that Ewing sarcoma (EWS) cells overexpress PARP and that modulation of PARP modified their sensitivity to apoptotic killing by drugs and ionizing radiation. Recent studies investigated the molecular mechanisms by which PARP participates in the apoptosis process. Current experiments relate to targeting PARP to further understand its roles and partners in regulating DNA repair and transcription to provide the rational for additional basic studies and potential clinical translational studies. These studies resulted from collaborations by Drs. Smulson, Dritschilo, Notario, Jung, and Soldatenkov, and represent a research area unique to the MTTR program and to Lombardi.

Top

Studies on pathway/target discovery and validation of estrogen-induced apoptosis
Investigators: Wellstein, Rosenthal, Riegel

This is a nationwide research program funded by the DOD as a Center of Excellence (COE) in its second year, with Dr. V. Craig Jordan as PI, who has recently been recruited to the BC Program at Lombardi from the Fox Chase Cancer Center. Other sites include the Fox Chase Cancer Center (Clinical PI: L. Goldstein) and TGen, Arizona (Genomics, PI: J. Trent). In the MTTR Program, one project of the COE is led by Dr. Wellstein (Proteomics and Pathway Integration). The COE has four Aims: (1) To identify protein signatures that are hallmarks of estrogen induced apoptosis in contrast to proliferation; (2) To decipher cellular signaling pathways and posttranslational modifications using proteomics; (3) To validate the apoptosis and survival pathways by integrating gene array and proteomic data, and; (4) To identify molecular drivers as novel targets for therapeutics. The discovery approach is as follows: Cells, tissue extracts from tumors (xenografts or human biopsies) or serum from patients are collected at FCCC before and after treatment with estradiol of distilbestrol (DES; in the clinical trial). At Lombardi, proteomics fractionation is carried out by immunoprecipitation (IP) or liquid chromatography (LC) followed by Mass Spectrometry (MS) analysis of the proteome. From the proteomics analysis we identify pathway alterations inducing apoptosis due to estradiol / DES treatment versus the induction of proliferation. Fractionation of the proteome from cells or tissues is done by IP for estrogen receptor-associated proteins, tyrosine phosphoprotein complexes and proteins complexed with nuclear receptor co-activators. The resulting data are analyzed at Lombardi in collaboration with the Protein Information Resource (PIR) that is housed at Georgetown University. Because Dr. Wellstein's broad range of research interests best place him in the MTTR program, this breast cancer-related research placed in this program and not in the Breast Cancer Program. Lombardi's involvement in this COE will likely increase as a result of Dr. Jordan's relocation to Lombardi.

These studies generated proteomics profiles under conditions of E2 induced growth or apoptosis and identified (de novo) differentially regulated or modified signaling proteins from cells harvested under different conditions and time points that are altered after an apoptotic response. Approximately 410 gel slices containing complexed proteins separated by gel electrophoresis were analyzed by mass spectrometry and protein sequencing. It was found that within 2 hours after E2 treatment, apoptosis is fully triggered as measured by the proteomics analysis. Amongst the phosphoproteins studied further, the nuclear receptor co-activator AIB1 (=SRC-3 or NCoA) activity was found to be controlled by tyrosine phosphorylation and detailed MS and mutational analysis revealed residue Y1357 as the most significant driver phosphorylation. pY1357 was increased in response to HER2/neu activation and is required to modulate AIB1/SRC-3 co-activation. c-Abl tyrosine kinase directly phosphorylates AIB1/SRC-3 and modulates the association of AIB1 with the transcriptional cofactor p300, and the methyltransferase CARM1. AIB1/SRC-3/NCoA dependent transcription and phenotypic changes can be reversed by the Abl kinase inhibitor imatinib. Thus, the phosphorylation state of Y1357 can function as a rate-limiting molecular on/off switch that facilitates the cross-talk between hormone, growth factor and intracellular kinase signaling. This work was published in Molecular and Cellular Biology in August 2008 (Oh et al, 2008) and points to abl or related kinases as potential targets to modulate AIB1 pathway activity that is a collaboration between Drs. Wellstein and Riegel (BC).

In future experiments the contribution of the other identified candidate signal proteins will be evaluated by molecular targeting (RNAi) or over-expression as well as detailed analysis of their posttranslational modifications and identification of modifying enzymes. These findings will be merged with mRNA data and siRNA library targeting initiated at the TGen site.

Top

Studies on the role of PI3K signaling in the response to radiation
Investigators: Waldman

Over the past few years, it was demonstrated that PTEN controls a radiation-induced cell size checkpoint and that PTEN can modulate p53 activation in human cells. In an effort to identify genes whose expression is regulated by activated phosphatidylinositol 3-kinase (PI3K) signaling, Waldman performed microarray analysis and subsequent quantitative reverse transcription-PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregulated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led to reversion of the gene expression program. Western blots revealed that p53 was stabilized in HCT116 PTEN(-/-) cells via an Akt1-dependent and p14(ARF)-independent mechanism. Stable depletion of PTEN in untransformed human fibroblasts and epithelial cells also led to up-regulation of p53 and senescence-like growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to continue proliferating. Additional experiments tested whether oncogenic PIK3CA, like inactivated PTEN, could activate p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally, targeted deletion of an endogenous allele of oncogenic, but not wild-type, PIK3CA in a human cancer cell line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies demonstrated that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on phosphatidylinositol (3,4,5)-triphosphate-induced mitogenesis during human cancer pathogenesis. Current work focuses on better understanding the mechanism of p53 activation by activated PI3K signaling as well as the role of PI3K signaling in modulating radiosensitivity using a glioblastoma cell system.

Top

Role of the PCPH oncogene in prostate cancer (PCa) invasion and resistance to chemoradiation
Investigators: Notario, Jorgensen

Previous reports showed that PCPH is mutated or dysregulated in some human tumors, suggesting a role in malignant progression. Immunohistochemical analyses showed that PCPH is not expressed in the normal prostate, but its expression increases during cancer progression from detectable in benign prostatic hyperplasia (BPH) to clearly expressed in prostatic intraepithelial neoplasia (PIN) and highly expressed in PCa. Investigation of the contribution of PCPH to the PCa malignant phenotype showed that forced PCPH over-expression in PC-3 cells increased collagen I expression and enhanced invasiveness, whereas shRNA-mediated PCPH knockdown in LNCaP cells, which express high PCPH levels, down-regulated collagen I expression and decreased invasiveness. PCPH regulated invasiveness and collagen I expression by a mechanism involving PKCδ: 1) PCPH knockdown in LNCaP cells decreased PKCδ levels relative to control cells; 2) PKCδ knockdown in LNCaP cells recapitulated all changes caused by PCPH knockdown, and; 3) forced expression of PKCδ in cells with knocked down PCPH reverted all changes provoked by PCPH down-regulation, and rescued the original phenotype of LNCaP cells. These results strongly suggested that the expression level and/or mutational status of PCPH contributes to determine the invasiveness of prostate cancer cells through a mechanism involving PKCδ. Data from immunohistochemical analyses in serial sections of normal, pre-malignant and malignant prostate specimens underscored the clinical significance of our findings by showing remarkably similar patterns of expression for PCPH and PKCδ, thus strongly suggesting their likely co-regulation in human tumors.

Understanding chemoradiation resistance mechanisms should facilitate the design of optimized therapy protocols against PCa. Recent studies revealed that PCPH confers resistance to cisplatin-induced apoptosis. PC-3 cells expressing a truncated, oncogenic PCPH variant protein (mt-PCPH) were found to be significantly more resistant to cisplatin-induced apoptosis when compared to PC-3 cells expressing the normal PCPH protein or to cells transfected with DNA of the corresponding empty vector as control. These and other findings suggest that targeting PCPH expression in combination with chemotherapy and/or radiation may improve the outcome of protocols currently used for PCa treatment.

Top

Targeting the pleiotrophin/anaplastic lymphoma kinase (PTN/ALK) pathway
Investigators: Wellstein

This is an example of inter-programmatic collaboration with Drs. Riegel  and Toretsky, which focuses on the growth factor pleiotrophin (PTN) and the ALK receptor both of which were found to be highly up-regulated in pancreatic adenocarcinomas, brain tumors, and in subsets of prostate, colorectal and breast cancers. Using an ELISA assay developed in the Wellstein laboratory, the ligand PTN was found to be increased in serum samples from patients with pancreatic and colorectal carcinoma in comparison to control subjects. Increased PTN and/or ALK expression levels in primary tumors were typically related to poorer disease outcome. More recently, ALK was described by others as being mutated and activated in familial neuroblastoma. Depletion of endogenous ALK or PTN mRNA demonstrates that either of them is rate-limiting for tumor growth in vivo of cells that express the receptor and/or the ligand. Based on this series of observations, the search for specific inhibitors of this pathway was initiated employing two independent approaches identifying: (1) antibodies to the ligand-binding domain (LBD) of the ALK receptor or to the ligand, and; (2) small molecule inhibitors of ALK.

Originally, the ligand binding domain (LBD) in the PTN-receptor was identified by phage display of cDNA libraries from fetal brain and placenta in which the PTN ligand was used as bait. Based on the discovery of the LBD of ALK, an ALK-GST fusion protein was produced, containing the LBD +/- 50 amino acids N-terminally and C-terminally for immunization of rabbits for polyclonal and of mice for monoclonal antibodies. In addition, a yeast two-hybrid screen was used for the identification of human single chain antibodies (scFv). The scFV were found to bind with high affinity (data obtained using the Lombardi Biacore Molecular Interaction shared resource) and specificity to the LBD as do mouse monoclonal antibodies raised against the LBD. Antibodies (monoclonal or scFv) that recognize the ALK LBD blocked PTN ligand binding and inhibited signaling. In addition, treatment of ALK+ tumor xenograft-bearing animal with these mAbs inhibited tumor growth of xenograft tumors. Analysis of the tumors showed apoptosis induction after antibody treatment of established tumors with the anti-ALK mouse IgG or induction of expression of scFv. In addition to xenograft models (MDA-MB231, U87, PC3 cells), the LSL-KrasG12D transgenic mouse model of pancreatic cancer progression was used. In this model, PanIN progression to invasive carcinoma occurs over the course of 6 months and ALK-targeted antibody treatment was used to assess its effect. Preliminary studies indicate that the treatment reduced the incidence of invasive carcinoma. In addition to these antibodies, small-molecule ALK kinase inhibitors were generated in collaboration with Dr. A. Kozikowski (formerly at Lombardi). Kinase assays showed several inhibitors with micromolar IC50, but none of them was more than 10-fold selective for the ALK kinase relative to the IGF1R or other closely related kinases, i.e., similar to UCNO1, a staurosporine derivative or AEW541, an IGF1R kinase inhibitor.

Future experiments will analyze a series of tumor types for inhibition by anti-ALK monoclonal antibodies in animals to decide on the appropriate tumor type to target for initial clinical studies. Also, several kinase inhibitor candidates from our screen and published by others are still being evaluated.

Top

Novel BRCA1-mimetic drugs for breast cancer
Investigators: Rosen, Tomita

The functional interaction between the breast cancer susceptibility gene-1 protein (BRCA1) and the estrogen receptor (ER-a) has been studied in Dr. Rosen's laboratory for the past 10 years. During these studies, it was observed that BRCA1 strongly inhibits the activity of ER-a (both ligand-dependent and ligand-independent) in breast carcinoma cells and blocks estrogen (E2)-stimulated gene expression and cell proliferation. Additional results showed that the BRCA1-mediated repression of ER-a activity is due to a physical interaction of BRCA1 and ER-a, which was mapped at a high level of resolution. Based on these data, a three-dimensional model of the BRCA1: ER-a interaction was generated and used to perform virtual screening of a small molecule library to identify compounds that might act as "BRCA1-mimetics" to insert deeply into ER-a at one of two key points of contact. Subsequently 40 such compounds were obtained and tested. Of the 40 compounds, six were found that strongly inhibited ER-a activity, including several that yield 50% inhibition at concentrations of only 2-4 mM. These small molecule drug-like compounds inhibit the activity of ER-a in both the liganded and unliganded state and at concentrations that do not cause non-specific cytotoxicity. And, importantly, they interact with the ER-a protein in a manner that is distinct from those of the selective estrogen receptor modulators (SERMs), such as Tamoxifen and Raloxifene. Thus they may ultimately be useful in breast cancer prevention as well as the treatment of a significant subset of hormone-resistant breast cancers that retain expression of ER-a. Future plans involve further studies and development of these compounds, including: 1) high resolution mechanistic studies of the interaction of the compounds with ER-a (in collaboration with Dr. York Tomita, 3DT); 2) chemical modification of the lead compounds to make them more potent and/or more "drug-like" (in collaboration with Dr. Milton Brown, 3DT); 3) preclinical toxicity and efficacy studies against estrogen-dependent human breast cancer xenografts in nude mice (in collaboration with Dr. Robert Clarke, BC), and; 4) breast cancer prevention studies in Brca1-deficient transgenic mice (in collaboration with Dr. Priscilla Furth).

Top

Radiation sensitization by inhibition of histone deacetylation
Investigators: Jung, Dritschilo

Recent emerging evidence supports the role of histone deacetylases (HDACs) in DNA damage response and radiation sensitivity. HDAC1, 2 and 3 are involved in the nuclear activation of NF-kB. ATM, which is postulated as an upstream kinase of NF-kB, has been implicated also in chromatin-based radiation responses. These observations also support HDAC inhibitors as sensitizers of cancer cells to ionizing radiation and provide insight into possible mechanisms for further investigation. Modification of proteins by histone acetyltransferases (HAT) or HDACs plays an important role in the control of gene expression, and its dysregulation has been linked to malignant transformation and other diseases. Although HDAC inhibitors have been extensively studied and several are currently in clinical trials, there is little information available on inhibitors of HATs (HATi). Using the natural HATi, anacardic acid, as the lead compound, a series of 28 analogues were synthesized and investigated for HAT-inhibitory properties and effects on cancer cell growth in collaboration with Dr. Milton Brown. The compounds inhibited up to 95% HAT activity in vitro, and there was a clear correlation between their inhibitory potency and cytotoxicity toward a broad panel of cancer cells. Interestingly, all tested compounds were relatively nontoxic to nonmalignant human cell lines. Western blot analysis of MCF7 breast carcinoma cells treated with HATi showed significant reduction in acetylation levels of histone H4. To directly show effect of the new compounds on HAT activity in vivo, MCF7 cells were cotransfected with the p21 promoter fused to firefly luciferase and a full-length p300 acetyltransferase, and luciferase activity was determined following treatment with HATi. Significant inhibition of p300 activity was detected after treatment with all tested compounds except one. Effects of the new HATi on protein acetylation and HAT activity in vivo make them suitable tools for discovery of molecular targets of HATs and, potentially, for development of new anticancer therapeutics. Current research focuses on investigating mechanisms underlying HDAC functions in intrinsic cellular radiation sensitivity as well as developing novel isoform-specific HDAC inhibitors in collaboration with Dr. Brown.

Top

Rapid minimally-invasive radiation biodosimetry through metabolomics
Investigators: Fornace

This project combines Dr. Fornace's metabolomics and stress-signaling expertise with the sensor-chip expertise of Sionex Corporation, to develop instrumentation for rapid non-invasive assessment of radiation exposure and injury using metabolic markers. Serum, urine, sweat, sebum, and saliva are the primary biofluids of interest. Gene expression profiling and the use of a genetic approach with mouse models will complement efforts focusing on human samples. Mouse models with disruption of relevant injury response pathways contribute to the delineation of injury signatures for key target tissues such as lymphoid, hematopoietic, and gastrointestinal injuries. To facilitate the processing of the samples, Lombardi has recently acquired a Waters UPLC Time-of-Flight Mass Spectrometry (QTOF) system. To date, 2000+ urine samples have been collected. The samples collected so far include two comprehensive dose response curves from 0 to 15 Gy and complete starvation (24 and 48hr) and lipopolysaccharide (LPS) studies. In addition to the collection of urine, the process of collecting saliva, serum and sebum samples is ongoing. Pilot studies have already begun in order to optimize the conditions for full identification of the metabolites and particularly of the more challenging non-polar molecules. Finally, expression profiling in blood from irradiated wild type mice is underway. Two radiation doses are being used at the moment, 2Gy and 8Gy, with considerable progress. Plans include expanding the dose range and comparing results with data from studies in patient blood. Genomic profiling will also be conducted in multiple tissues with various radiation doses and will also include the mouse models used in the metabolic profiling.

Top

Interaction with Clinical Radiation Oncology
Investigators: Gagnon, Collins, Collins, Harter, Dritschilo

While Lombardi has not been active in multi-center clinical trials through the RTOG and related groups, several changes have taken place following the arrival of the new Director to foster clinical research in radiation oncology. Lombardi is now supporting: 1) radiation therapy-based clinical research through the Clinical Resarch Management Office (CRMO), and; 2) selected high-priority clinical studies to examine CyberKnife treatment in conjunction with chemotherapy. The CyberKnife is a radiosurgery device consisting of a robotic arm that directs the pointing of an x-band linear accelerator. The central benefit of this treatment device is the robotic gantry, which allows a wide variety of radiation beams from a large solid angle to target with a great deal of accuracy. The removal of the geometric restrictions of a single jointed gantry, common in conventional accelerators, also allows non-isocentric treatment and true 3-dimensional respiratory tracking, giving highly conformal and homogenous dose-plans with relative ease of delivery. The radiation medicine department has broad experience with this technology and can be considered to have an international reputation within the radiosurgery community.

Several IRB-approved protocols utilizing the CyberKnife are accruing as of April 1, 2009:

  • A partial-prostate dose escalation study for prostate cancer patients after conventional radiation treatment. This study seeks to determine if single fraction prostate boost can be given to a sub-volume of the prostate harboring malignancy safely, and what dose can be given - a dose-escalation study. The partial-prostate volume is determined by probability plots of tumor distribution on AFIP prostate resection specimens compiled by the ISIS radiology group at Georgetown. To date, 5 patients have been entered into this protocol.
  • A prostate CyberKnife monotherapy protocol. This utilizes 5 fractions of CyberKnife treatment targeted to the entire prostate. This requires dose-homogeneity within the prostate. Eligible patients have low or intermediate risk tumors. Seven patients have been enrolled on this protocol.
  • A CyberKnife high dose radiation-like protocol where three fractions of CyberKnife are given after 5 weeks of conventional radiation. This study is based on the available data using brachytherapy for the three-fraction boost. The conventional external beam radiation is expected to sterilize microscopic periprostatic tumor extentions and the boost is expected to capitalize on the dose-fraction sensitivity of prostate cancers. Four patients have been entered on this protocol.
  • A study evaluating the outcomes of CyberKnife treatment of early stage non-small cell lung cancer and solitary pulmonary metastases. Three large radiation doses are delivered to the target with minimal margin in medically or technically inoperable patients. Two patients have been entered onto this study.
  • A dose-escalation study of CyberKnife treatment for primary or metastatic liver tumors. This study seeks to determine the maximally tolerated dose delivered in three fractions for patients with inoperable tumors. One patient has been entered onto this study.
  • A prospective evaluation of pain and quality of life outcomes in patients treated to the spine using Cyberknife radiosurgery. Data collected from 200 patients have formed the basis for several publications.
  • In collaboration with Dr. Fornace's lab, Drs. Gagnon and B. Collins are undertaking a study of metabolomic profiling of patients prior to and after conventional beam low-does radiation and CyberKnife high-dose radiation. The strategy is to develop metabolomic signatures of radiation exposure with a combination of ultra-pressure liquid chromatography (UPLC) coupled with highly sensitive time-of-flight (ToF) mass spectrometry (MS) to profile small molecules (<1 kDa), using the Proteomics and Metabolomics Shared Resource (PMSR). As the two radiation treatments differ in that large volumes are treated with conventional radiation with low-dose fractions while small volumes are treated with the CyberKnife with high dose-fractions. The group hopes to determine if a metabolomic signature is present from radiation and, ultimately, if different signatures can be elucidated for the different radiation techniques. Eight patients have been accrued so far.

Several protocols, pending IRB approval, employ the CyberKnife as a significant component of treatment:

  • A study of CyberKnife Radiogenic Therapy of Central Lung Tumors Using the TNFeradeTM Adenoviral Vector Protocol. Tumor Necrosis Factor-α (TNFα) is a potent multifunctional cytokine with well-demonstrated anti-cancer activity in both in vivo models and human clinical trials. Unfortunately, the systemic toxicity of this cytokine is a serious limitation to its use. One approach to limiting the systemic effects is through a gene therapy approach whereby the human TNFα cDNA is ligated to Egr-1, a radiation-inducible promoter. This promoter is further enhanced by multiplication of the "CArG" elements, responsible for the radioinducibility of the promoter gene. The construct is packaged in a replication-deficient human adenovirus to form TNFerade (GenVec, Inc., Rockville, Maryland). After intratumoral injection, TNFα expression can be induced by ionizing radiation in a dose-dependent manner. The superb spatial dose distribution achievable with the Cyberknife is expected to limit the effects of this potent cytokine to the high-dose radiosurgery volume. There is also probable synergy between the lethal effects of TNFα and radiation (Hallahan DE. Proc Natl Acad Sci USA 1989;86:10104-10107). The proposed protocol, for which grant support has been awarded, is a phase I/II radiation dose-escalation trial for central lung tumors, escalating from 8 Gy x 3 fractions to 14 Gy x 3 fractions, with CyberKnife treatment beginning the same day after a single intratumoral injection of TNFeradeTM via bronchoscopy. Measurements of serum TNFα in the peri-treatment period and follow-up with CT, CT/PET, and PFTs are planned.
  • A CyberKnife trial of pre-operative treatment for pancreatic adenocarcinoma. Higher radiation doses and a shorter treatment course might improve on results of pre-operative XRT protocols and improve respectability, while tighter dose-conformity might limit risk of operating in irradiated tissue. 500 cGy x 5 is planned followed by surgical exploration for possible resection.

Top

Functional regulation of base excision repair (BER) and nucleotide excision repair (NER) pathways in mammalian cells
Investigators: Roy

Dr. Roy is the PI of a 5-year R01 grant from NCI to study the mechanisms in DNA repair pathways for alkylation and deamination damage induced endogenously or from exogenous exposures, such as tobacco-specific nitrosamines, vinyl chloride, or alkylating chemotherapeutic agents. The studies include molecular mechanisms of catalytic activity of N-methyl purine DNA-glycosylase (MPG) in vitro and in vivo and the regulation of the latter by protein-protein interactions. His lab investigates how the DNA glycosylases such as MPG, a multisubstrate enzyme that initiates the base excision repair (BER) pathway for etheno, alkylated and deaminated bases, can locate or discriminate damaged bases in the vast ocean of normal bases. They found that MPG bound to base-pair mismatches especially to thermodynamically less stable pyrimidine-pyrimidine pairs, without catalyzing detectable base cleavage and concluded that the initial recognition of substrate base lesions by MPG is dependent on the ease of flipping of the base from unstable pairs to flexible catalytic pocket. Dr. Roy's ongoing studies show that the MPGs and other BER initiating enzymes' substrate recognition is regulated by multiple factors; in addition to thermodynamic stability and the dominantly ionic interaction at the lesion site on DNA and Mg2+, its N-terminal tail, which is indispensable for its catalytic activity, and other protein factors like AP-endonuclease (APE), the enzyme next to MPG in BER pathway, and BRCA1. Furthermore, MPG-mediated repair is slow, but predominant in the G0/G1 phase of the cell cycle in colon cancer cells. These studies are now being advanced with Drs. Toretsky, Uren  and Rosen. Dr. Roy also is a PI of another R01 grant to study the role of repair of oxidized bases in hepatocellular carcinoma (HCC) using the Long-Evans Cinnamon (LEC) rat, a useful animal model in studying mechanisms of spontaneous carcinogenesis induced by oxidative stress. His group found that both the activity and expression at the protein and RNA levels of major BER DNA glycosylases, endonuclease III (NTH1) and 8-oxoguanine DNA-glycosylase (OGG1), which initiate the excision and repair of oxidized bases, were significantly altered during the acute and early chronic phases of hepatitis induced by copper accumulation in the liver prior to the appearance of preneoplastic foci in the liver.

The above findings correlate with elevation of mutagenic DNA damage in the inflamed liver and show for the first time in an animal model that the repair of endogenous DNA adducts plays a critical role in the development of spontaneous HCC following chronic inflammation. Thus, suppression of DNA repair functional activity may be a risk factor for HCC. Ongoing studies in the project involve extensive inter-programmatic collaboration with Drs. Chung, Ressom, Feng, and Goldman in prostate and lung cancer research, where an R21 application has already been submitted and other proposals are being developed by this group. Dr. Roy also is now extending their DNA repair functional activity and single nucleotide polymorphism (genotype) assays into human populations to test their efficacy as biomarkers in human populations with HCC risk in collaboration with Drs. Loffredo and Goldman.

Top

Role of BRCA1 in the base excision repair (BER) pathway
Investigators: Rosen, Roy

The BER pathway is the major mechanism for the repair of damage to DNA bases caused by reactive oxygen species (ROS), a process that is thought to contribute significantly to the development of age-related pathologies, including cancer. BER is a high fidelity repair process that is both anti-mutagenic and anti-carcinogenic. Recently, Dr. Rosen's laboratory, in collaboration with Dr. Roy, has shown that BRCA1 stimulates several early steps in the BER pathway, including those involving enzymes that repair specific types of oxidative DNA lesions: 8-oxoguanine (repaired through the DNA glycosylase OGG1); thymine glycol (repaired through the DNA glycosylase NTH1), an abasic lesion (repaired through the apurinic/apyrimidinic endonuclease APE1). Thus, BRCA1 significantly enhances the ability of cells to repair these potentially mutagenic DNA lesions due to ROS and other genotoxic agents. These studies represent a significant advance in understanding the tumor suppressor function of BRCA1. They may also have practical consequences, because they suggest that individuals with germ-line BRCA1 mutations may be defective in this pathway, and this defect could potentially be ameliorated by the use of antioxidants. In addition, it becomes possible to stimulate BRCA1 activity and expression by the use of certain phytochemical agents such as indole-3-carbinol (I3C), its metabolite diindolylmethane (DIM), and the soy isoflavone genistein.

Top

Mouse models for estimates of intestinal tumorigenesis by space radiation
Investigators: Fornace

Ionizing radiation is a known risk factor for colorectal (CR) cancer. Considering the high incidence of gastrointestinal (GI) cancer in the US and even higher incidence of pre-malignant lesions, such as colonic polyps, even a modest increase by radiation exposure could have a significant effect on health risk estimates for manned space flight. During space travel, astronauts are exposed to high levels of radiation, which increases their risk for acute short-term radiation effects and for long-term deleterious effects like cancer. Long duration space missions, like those at the International Space Station, lunar missions, or a mission to Mars, increase astronauts' exposure to space radiation and the risks associated with such exposure. This project is important because there are no good estimates for intestinal, particularly CR, cancer by space radiation. The plan is to apply the well-characterized APC+/min mouse model of intestinal tumorigenesis to develop risks estimates for various radiation exposures during manned space flight. To undertake a comparative study of intestinal tumor risk in mice following exposure to different types of radiation, ongoing experiments are designed to determine the relative toxicity of 56Fe (iron) particles and protons relative to γ-rays, to allow standardization with doses equitoxic to 2 and 5 Gy of γ-radiation in C57Bl/6J mice, because acute toxicity parameters for the planned radiation sources is not available, while the corresponding γ-ray doses have been shown to increase intestinal tumorigenesis.

Two runs in the first year of this project at the NASA space radiation research laboratory (NSRL) at Brookhaven National Laboratory (BNL) have been already conducted. Results to date indicate that the relative biological effectiveness (RBE) for 56Fe particles compared to γ-rays is 1.29. This RBE is surprisingly low and is an important parameter that will allow us to plan long-term experiments. Irradiated mice are being observed long-term, and specific tissues are also being provided to various NASA investigators to complement their own projects. In the second run (Sept 2007) Fornace and colleagues also irradiated APC+/min mice with iron particles and protons. A total of 20 mice were irradiated and are being monitored for intestinal tumorigenesis. While not statistically significant, it is interesting that a few irradiated mice developed high intestinal tumor load within the first 100 days after irradiation, while this was not seen in unirradiated mice. At the end of the observation period (100-150 days), mice will be euthanized for intestinal tumor counting, as well as for genetic and molecular pathway analyses of individual tumors.

Top

Radioprotection by DIM
Investigators: Rosen, Dritschilo

A potentially important discovery has been made recently: that DIM (diindolylmethane), the major active metabolite of indole-3-carbinol, a cancer prevention agent derived from cruciferous vegetables, is a strong radioprotector against ionizing radiation (g-rays). It is particularly interesting that the protection is principally due to a broadening of shoulder of the radiation survival curve, since this occurs at lower doses in the range of the dose fraction size utilized in clinical radiation therapy and smaller. Importantly, strong protection is observed at low physiological concentrations of DIM (1 µM and less). The protection requires BRCA1 where it is abrogated by knockdown of BRCA1 and it is not observed in BRCA1-deficient cells. More specifically, the protection requires phosphorylation of BRCA1 on serine-1387. The radioprotection can also be abrogated by ATM and ATR, upstream DNA damage-responsive kinases that mediate BRCA1 phosphorylation in response to ionizing radiation. Additional data further showed that DIM rapidly induces phosphorylation of BRCA1 on serine-1387 in an ATM-dependent manner. At present, studies are underway to further delineate the mechanism of DIM radioprotection and evaluate its significance. Two potential applications for this research are being taken into consideration: 1) as a means of radioprotection in the setting of radiation exposures to the normal population (DIM is already under clinical use in chemoprevention studies); and 2) radioprotection of normal tissue in the setting of cancer treatment. In the latter case, although DIM protects both normal and cancer cell types, it does not protect BRCA1-deficient cells; and thus it might be useful to protect normal tissues in patients with BRCA1-deficient tumors (e.g., a subset of breast and ovarian cancers). Finally, Dr. Milton Brown has synthesized about 40 derivatives of DIM. Testing of these DIM derivatives is underway to identify compounds that may be even more efficacious as radioprotectors than DIM.

Top